Ethics
The trial was performed abiding by the Declaration of Helsinki [7], Good clinical practice (GCP) [8] and the guidelines of China National Medical Products Administration (NMPA). Relevant documents, including protocol, informed consent and drug inspection report were all approved independently by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University. All protocol violations occurred have been reported to the Medical Ethics Committee.
Subjects
The inclusion criteria for the volunteers included as follows: 1) Healthy male or female aged 18 and above. 2) The body mass index is in the range of 18.6-28.5 kg/m2 (including the critical value). The weight of male is not less than 50.0 kg, and that of female is not less than 45.0 kg. 3) The following examination show that the indicators are normal or abnormal without clinical significance. The examination including: Vital signs, physical examination, blood routine, blood biochemistry, urinalysis, pregnancy test for female, serological tests for hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), and syphilis virus, 12 lead ECG, breath test for alcohol, drug abuse test. 4) The subjects have no family planning within 3 months and could select contraceptive method. 5) Before the study, all subjects have been informed of the study’s purpose, protocal, benefits, and risks, and signed the informed consent voluntarily.
The exclusion criteria for the volunteers included as follows: Being allergy to the study medications, smoking, alcohol abuse; and participation in another clinical trial within 3 months.
Study design
The single-dose randomized, open-label, two-period crossover study was executed in the Phase I Clinical Research Center of the Affiliated Hospital of Qingdao University. According to the random table generate by SAS 9.4, the subjects were divided into two groups at the ratio of 1:1 (Table 1). The select qualified volunteers were hospitalized in the Phase I Clinical Research Center, and fasted for 10 hours overnight until administration. The medicine was swallowed with 240 ml water at room temperature. Blood samples were taken before administration and at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 36, 48, 72, 96, 120, 144, 168 hours after administration. The samples were centrifuged at 1,800 g for 10 min at 4 °C to separate the plasma. The plasma samples were divided into two aliquots and stored at -80 °C until bioanalysis. The half-life of levamlodipine is 30 ~ 50 hours. Washout period, the interval between two administration, is 21 days. In the two periods, the operation was kept the same.
Moreover, in high fat meal group, the high-fat breakfast was arranged within half an hour before taking the medicine. Other procedures were the same as those in the fasting group.
Safety assessment
The safety of levamlodipine besylate tablets was assessed by monitoring vital signs and laboratory tests. Vital signs, such as body temperature, blood pressure, and heart rate, were measured before administration and at 3, 8, 24, 36, 48, 72, 96, 120, 144, 168 hours after that. Before discharge, the subjects were evaluated with blood routine, blood biochemistry, urinalysis, pregnancy test for female, and 12 lead ECG. For adverse events (AEs), clinical symptoms, severity, occurrence and ending time, duration, treatment measures and the correlation with the drugs were recorded. All of the AEs that occur within the 7 half lives of the drug (a half-life 30-50 hours of levamlodipine) were recorded and followed up, unless the subjects returned to normal or stable, or failed to visit.
Bioanalysis
The analysts were blinded to the randomization. Plasma samples were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was tested by Suzhou Shenglin Pharmaceutical Technology Co., Ltd. An ACQUITY ultra-high-performance liquid chromatography unit (SHIMADZU, Nexera UHPLC LC-30A, Japan) and a mass spectrometer (Applied Biosystems, MDS Sciex, Triple Quad 6500 plus, Concord, Canada) was used in the study. Under multiple reaction monitoring, LC-MS/MS system adopts positive ionization mode. Data collection and handling was employed with Analyst 1.6.3 software (Applied Biosystems, Foster City, CA, U.S.A.). The solid-phase extraction (SPE) experiments [9] were performed by HLB 96-well Plate (Waters Oasis, WAT058951). The plate contains a reversed phase functionalized polymeric sorbent (30 mg/well), in which particle size is 30 μm. Scheme 1 summarized several steps about the cleaning and extraction process. (a) The cartridges were activated by 800 μL of methanol and 800 μL of water followed. (b) The column was loaded with 150 μL of plasma sample or calibrator, 50 μL of internal standard working fluid (5 ng/mL) and 100 μL of deionized water. (c) 500 μL of deionized water twice and methanol (80%) followed were forced to pass through the column to to achieve depolarization. (d) Levamlodipine was eluted from the column by 500 μL of pure acetonitrile twice. Then the solution was blown dry by a pure N2 stream. 150 μL of pure acetonitrile was add into each sample, and mixed at room temperature for 10 minutes. Ultimately, 20 μL of the extracted sample was injected into the LC MS/MS system.
The method is verified fully by selectivity, accuracy, precision, calibration curve and stability. The drug concentration was linear in the range of 0.0500 ~ 10.0 μg × L–1. The lower limit of quantification was 0.05 μg × L–1, and the standard curve was Y = 0.55667X - 0.0030182 (r2 = 0.9949). The intra- and interday maximum precision was 5.4% and 4.8%, respectively. The intra- and interday accuracy was -6.7 ~ 3.9% and -3.3 ~ 3.3%, respectively. The extraction recovery of levamlodipine was 94.7 ± 3.9%. There was no significant interference in selectivity or stability.
Pharmacokinetic analysis
All subjects completed the study and were included in the pharmacokinetic analysis. The pharmacokinetic parameters were calculated according to non-compartment model using Phoenix™ WinNonlin® 8.0 software (Pharsight, St. Louis, MO, USA). The value below the lower limit of quantification that occur before the first measurable concentration are set as zero. Subsequent values below the lower limit of quantification were excluded. Linear trapezoidal rule is applied in AUC0-t calculation. AUC0-∞ is the sum of AUC0-t and the ratio of the last measurable concentration (Ct) to the elimination rate constant (Ke). The maximum plasma concentration (Cmax) and the time to Cmax (Tmax) can be obtained from the concentration-time curve. Elimination half-life, T1/2 = 0.693/Ke.
Statistical analysis
Analysis of variance (ANOVA) was performed on the ln-transformed Cmax, AUC0-t, and AUC0-∞ to assess the effects from subject, treatment, period, and preparation. Statistical data were presented as mean ± standard deviation (SD). The probability value less than 0.05 is considered statistically significant. Calculate the 90% confidence intervals (CIs) of the geometric mean ratio of the main indicators. If it is within the equivalent range (80 ~ 125%), it is judged as bioequivalence, and the results of double unilateral t-test are listed. Tmax was analyzed by non-parametric statistical test. Statistical analyses were performed by SAS 9.4 (SAS Institute Inc. Cary, NC, USA).